1. Introduction
Innate immunity represents an early defense system in vertebrate and invertebrate animals [
1]. It involves physical barriers, phagocytic cells and a variety of proteins which include recognition and effector molecules, that coordinately act to block infections and promote inflammation as a further defense mechanism [
1]. One of the most ancestral innate defense is encapsulation, a process referring to the binding of multiple cells to a large invader, that cannot be phagocytized by a single cell [
2,
3]. This process begins soon after the recognition of the invader by cellular sensors of pathogen-associated molecular patterns (PAMPs) and it is characterized by abundant production of reactive oxygen species (ROS), thus facilitating the elimination of the intruder [
3]. In arthropods, the production of a capsule surrounding the invader is followed by its melanization to enclose the invader into an environment with high concentration of ROS [
3]. Accordingly, the melanin’s ancestral role was assumed to be linked to this defense mechanism [
4], even if melanization is also involved in other biological processes like wound healing, clot formation, pigmentation and UV-protection [
5,
6].
Although melanotic encapsulation is mostly described in arthropods [
7], the presence of dark, melanin-like granules surrounding pathogens has also been reported in several mollusk species. Examples for this come from the gastropod
Achatina fulica infected by nematodes [
8],
Roseovarius crassostreae infecting
Crassostrea virginica and
Vibrio tapetis infecting
Ruditapes phlilippinarum [
9]. Here, all pallial epithelia of bivalves were capable of melanization [
9], but most of the genes involved in the melanogenic pathway were highly expressed in the mantle edge [
10,
11], a tissue involved in both defense mechanisms and shell formation in bivalve and gastropod mollusks [
12,
13,
14].
Melanin originates from tyrosine and related phenolic compounds, which are hydroxylated into
o-diphenols, oxidized into quinones and polymerized to obtain melanin [
15]. Although most of the gene complements of the melanogenic pathways have been traced in vertebrates and arthropods [
16,
17], the enzyme functions promoting the last enzymatic reaction (EC 5.3.3.12), namely the conversion of dopachrome into 5,6-dihydroxyindole (DHI) or 5,6-dihydroxyindole-2-carboxylic acid (DHICA) [
18], were seldom investigated. This enzyme, called dopachrome tautomerase (DCT) [
15], was initially discovered in mice in 1980 [
19]. Nowadays, it is known that human DCT regulates the production of ROS facilitating human papillomavirus infection [
20], it is involved in the progression of melanomas [
21] and it protects melanocytic cells from ROS damage [
22]. Subsequent to DCT, an enzyme with an equivalent function was characterized in the insect
Manduca sexta [
23]. This gene, known as dopachrome decarboxylase/tautomerase or dopachrome converting enzyme (DCE), was previously discovered in
Drosophila melanogaster and called
yellow. It was reported associated with larval pigmentation defects [
24] and the yellowish pattern of defectives flies was only later explained by transposon-mediated mutagenesis and altered melanization enzyme activity [
25]. Although all the members of the
yellow gene family shared a conserved major royal jelly protein (MRJP) domain, not all
yellow proteins can convert dopachrome into DHI, like
D. melanogaster yellow-f2 and
Aedes aegypti yellow [
26,
27,
28]. In the honey bee,
yellow proteins are the main component (80%) of the royal jelly [
29]. In addition to protective and antimicrobial functions, royal jelly is responsible of the larval development into queens [
30], although the function of MRJPs in this process has yet to be elucidated [
29,
31].
Yellow genes have been reported as a gene family taxonomically restricted to insects, likely originating from horizontal gene transfer (HGT) from bacteria, and functionally diversified by species-specific duplications [
32]. For instance,
yellow-like transcripts found in the venom of a parasitoid wasp modulate host physiology and behavior [
33],
yellow-like salivary proteins of pathogen vectors, such as the sand flies
Phlebotomus tobbi and
P. sergenti, displayed leishmanicidal properties [
34,
35] and
yellow proteins of the leaf beetle
Phaedon cochleariae likely protect larvae from fungal infections [
36]. Other
yellow-like genes were identified in fungi and micro-eukaryotes, but their roles remains to be determined [
37]. Apart from the aforementioned microscopic organisms and insects,
yellow-like genes have been reported in few other species, namely in
Brachiostoma floridae (Chordata), in the amoeba
Naegleria gruberi and in the copepod
Lepeophtheirus salmonis [
32].
In bivalves, the enzymes involved in the biogenesis of melanin are often grouped into the general class of phenoloxidases, including three distinct enzyme types: tyrosinases (EC 1.14.18.1), catecholases (EC 1.10.3.1) and laccases (EC 1.10.3.2) [
38], whereas less is known about the existence and functional role of mollusk dopachrome tautomerase genes.
The steady increase of sequenced genomes provides the opportunity for a comprehensive analysis of dopachrome tautomerase enzymes in metazoans. In this paper we investigated the taxonomic distribution as well as the phylogenetic relationships of DCT and DCE/yellow genes in genomic resources from a wider taxonomic distribution with a special focus on the lophotrochozoan phyla. By combining genome sequences with transcriptomic data, we could further demonstrate that these genes are functional and probably also underwent functional diversification outside of insects.
4. Discussion
Melanin is widespread in the animal kingdom, where it is involved in several important biological functions including pigmentation and immune-defense [
6,
8,
66]. Here, we now traced the distribution of the DCT and DCE/
yellow gene families through metazoan evolution. DCTs possess the capacity to convert dopachrome into DHICA [
17,
18], while some DCE/
yellow proteins can convert dopachrome into DHI. Both DHICA and DHI, combined in different ratios, are needed to build eumelanin [
6,
15], and their enzymatic conversion is considered to be the bottleneck of melanin production [
19].
Both gene families appeared as highly dynamic, whose distribution is characterized by extensive gene losses and, possibly, horizontal gene transfer (HGT) events. We showed that DCTs are mainly found in deuterostomes and only in a few non-deuterostome organisms, including cephalopods, platyhelminths, and
C. teleta as only representative of nematodes (
Table 1). As eumelanin represents the dark pigment in cephalopod ink [
6] and in the black eye spots of platyhelminth species [
67], DCTs seem to play a crucial role for dark coloration in a wide range of taxa. However, eumelanin is also widespread in insects and other invertebrates [
17], most of which do not encode a DCT gene. Indeed, melanization is a normal process during shell formation in mollusks [
68].
This suggests that the enzymatic function of DCT (dopachrome tautomerase) is performed by a different protein, with DCE/
yellow being a likely candidate [
17]. The DCE/
yellow family was previously thought to be exclusively found in insects, bacteria and fungi [
29,
32]. Our results now suggest that along with the early-diverging metazoan phyla of Placozoa, Porifera, Cnidaria, and Ctenophora, only the lophotrochozoans Annelida, Nemertea and Phoronids do not possess these genes. In contrast, we found that almost all other analyzed lophotrochozoans have one or more DCE/
yellow genes, while cephalopods and lancelets even possess both DCT and DCE/
yellow genes (
Table 1).
This patchy distribution suggests that DCT and especially DCE/
yellow genes were already present in the protostome and deuterostome ancestor and both genes could have experienced extensive gene loss events. DCE/
yellow was then lost in deuterostomes, in Malacostraca among arthropods as well as in the lophotrochozoan phyla mentioned above, whereas DCT has been lost in most protostomes, with the exception of cephalopods. The presence of a DCT gene in a single nematode species (
C. teleta), as well as the presence of DCE/
yellow genes in lancelets can possibly be explained by HGT events. Prokaryote-to-Eukaryotic HGT is considered to be an important element driving eukaryote genome evolution [
69,
70], whereas Eukaryote-to-Eukaryote HGTs were only seldom reported (e.g., in the foraminifera
R. filosa [
71]). Curiously, in the phylogenetic tree DCE/
yellow proteins of lancelets were closely related to the DCE/
yellow of
R. filosa, possibly indicating an HGT event (
Figure 3).
This extensive gene loss scenario might be feasible due to the functional redundancy of DCT and DCE with either gene being able to compensate the loss of the other in the production of melanin. Similarly, both
D-dopachrome tautomerase (D-DT) and its paralogue
Macrophage migration inhibitory factor (MIF) also possess a similar enzymatic capacity, although on the non-physiological substrate D-dopachrome [
72,
73]. When honey bee
Major Royal Jelly Protein 1 is artificially expressed in mice the production of D-DT is decreased [
74], indicating a possible potential compensative role between these two proteins. We recently investigated the taxonomic distribution of D-DT genes demonstrating a patchy distribution among protostomes, with gene losses in insect and crustacea as well as in the bivalve
C. gigas ([
75] unpublished data until accepted). Although the hypothesis that DCT, DCE/
yellow and D-DT gene losses could be functionally compensated, this hypothesis requires experimental proof and, on the basis of available knowledge, it remains a speculation.
The presence of a gene or conserved domain does however not demonstrate that it is functional. One line of evidence that might support functionality comes from the expression of non-insect DCE/
yellow genes. Although we retrieved abundant RNA-seq datasets for some mollusk species, DCE/
yellow expression levels show often limited to very limited expression values (
Figure 5). Among bivalves, we showed a preferential of
C. gigas DCE/
yellow expression in gills and mantle tightly timed with ontogeny (
Figure 6). DCE/
yellow expression was also substantially down regulated in the oyster gills after OsHV-1 infection suggesting a virus-mediated modulation of the host’s DCE/
yellow pathway. This would support that one of the pleiotropic functions exerted by insect DCE/
yellow proteins is the modulation of host-parasite interactions [
34,
64]. In this context, the
Mytilus-
Mytilicola host-parasite system represents the only situation in which both host and parasite encode a DCE/
yellow gene. While the mussel host did hardly express its DCE/
yellow gene in the gut, the gene of the parasitic copepod was among the most abundant transcripts (
Figure 5). Such high expression level possibly represent a response to the challenges posed by the gut environment and to the release of ROS by the mussel host [
60], an hypothesis consistent with the known ROS scavenger function of melanin [
66]. In the free-swimming copepod
T. kingsejongensis as well as in all developmental stages of the ectoparasitic salmon louse
L. salmonis, on the other hand, we found only low expression levels of their DCE/
yellow genes further suggesting species-specific functional diversification and a prominent role of DCE/
yellow proteins limited to the
Mytilus-
Mytilicola host-parasite interaction. Arguably, also the salivary MIF of ticks and aphids are involved in the modulation of host-pathogen interactions [
76], intriguingly mirroring the role of insect’ salivary yellows [
34,
35,
64]. Therefore, functional validations are definitively needed.
The active expression of
B. belcheri DCE/
yellow genes also suggests that it serves a functional role although this gene might have originated from HGT. Finally, we found that in
O. bimaculoides as one of the few species possessing both DCT and DCE/
yellow genes, DCT is exclusively expressed in the retina and DCE/
yellow in the skin tissues. In humans, both these tissues showed high expression levels of DCT [
77,
78] and this intriguing similarity might suggest that both octopus DCT and DCE/
yellow genes serve functional roles in different tissues. Structural modelling of both octopus and lancelet DCE/
yellow proteins revealed an overall structural similarity with insect’s
yellow, although the conservation of key residues located inside the putative enzymatic pocket is low. This latter result, in particular for the octopus DCE/
yellow protein is striking, since the high expression in skin tissues strongly suggests its involvement in the melanogenic pathway. Interestingly, both these two proteins showed a conserved aromatic residue, that is also present in a putative hydrolase protein of the bacterium
A. variabilis, further support the connection between bacterial and metazoan DCE/
yellow genes.
Ferguson and collaborators [
32] proposed that the metazoan
yellow gene family originated from an HGT from bacteria. However, previous phylogenetic analysis failed to identify a clear link between bacteria and insect
yellow genes, since the co-clustering of bacterial and of
yellow-x hits, the most ancestral clade of insect’s
yellows, greatly depended by the parameters adopted for the analysis [
32,
63]. We are aware that, also in our analysis, the high heterogeneity of DCE/
yellow genes, coupled with the small number of informative sites, somewhat limited the resolution of the phylogenetic tree. However, by increasing the number of the originally sampled species, we showed a clear distinction between the DCE/
yellow sequences of the different animal phyla (
Figure 3). Among the cluster of each phylum, the DCE/
yellow sequences only partially followed the phylogenetic relationships among species, indicating the presence of different functional subtypes shared between species of the same phyla. This aspect is particularly evident for insect genes, where this gene family underwent a rapid evolution, resulting in high sequence diversification and remarkably different functional adaptations between and within species [
26,
32,
63]. Likewise, we reported that also mollusk DCE/
yellow genes experienced considerable diversification in some species, like in
M. yessoensis (11 genes), in Mytiloida species (3–5 genes) and in gastropod species (
Haliotis spp. and
B. glabrata). Although we could trace one intronless DCE/
yellow in several lophotrochozoan species, we observed a considerable syntenic conservation only for species of the same family (e.g., Ostreoida,
Supplementary File 2). Low conservation of the flanking genes was also reported for insect DCE/
yellow, including the low conservation of their cys-regulatory landscape [
79], further supporting the dynamic nature of evolution in this gene family.
The peculiar DCE/
yellow protein structure of ostreid bivalves nicely illustrates these evolutionary dynamics. In oysters DCE/
yellow is composed of two MRJP domains (
Supplementary File 3), and the high similarity of these two MRJP domains, along with the lower conservation of key residues as well as of the overall structural features possibly indicate a recent incomplete duplication followed by functional liberation and random drift through mutational and functional space. Moreover, the presence of marked structural differences of the
C. gigas protein, taken as example for bivalve DCE/
yellow proteins, can suggest that these genes have evolved different functions starting from the same structural core (
Figure 4).
This structural gene diversification might suggest that melanin production is not the only function served by DCE/
yellow genes. Indeed, while all DCTs can convert dopachrome into DHICA [
17,
18], only a subset of the genes described as DCE/
yellow can convert dopachrome into DHI. Other DCE/
yellow genes are involved in cuticle and eggshell formation and hardening during insect development, protection from dehydration and, as salivary proteins, they confer protective immunity against
Leishmania major as well as they can bind several ligands, including serotonin [
26,
29,
32,
63,
64]. The structural analyses of the
M. intestinalis DCE/
yellow protein revealed shared common features with
D. melanogaster DCE, and such features were also present in the salivary
yellow proteins of
Phlebotomus but not in the
A. mellifera MRJP structural model (
Figure 4). Given these similarities, we could be tempted to speculate that these two proteins may retain an activity similar to that of salivary proteins, but in-vitro experiments with recombinant proteins are needed to demonstrate these functions.